胰岛素样生长因子2信使RNA结合蛋白3对弥漫型胃癌细胞增殖、迁移和M2巨噬细胞极化的影响  

Effect of insulin-like growth factor-2 mRNA-binding protein 3 on proliferation, migration of diffuse gastric cancer cells, and M2 macrophage polarization

作　　者：朱天宇 洪莲莲 凌志强 Zhu Tianyu;Hong Lianlian;Ling Zhiqiang(Department of Oncology,the Second Clinical Medical College of Wenzhou Medicine University,Wenzhou 325027,China;Zhejiang Cancer Institute,Zhejiang Cancer Hospital,Hangzhou 310022,China;Hangzhou Institute of Medicine Chinese Academy of Sciences,Hangzhou 310018,China)

机构地区：[1]温州医科大学第二临床医学院肿瘤科,温州325027 [2]浙江省肿瘤研究所,浙江省肿瘤医院,杭州310022 [3]中国科学院杭州医学研究所,杭州310018

出　　处：《中华实验外科杂志》2024年第5期969-973,共5页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金面上项目(32271238);浙江省自然科学基金面上项目(LY20H160005);浙江省卫生领军人才(浙卫办[2021]40号);浙江省公益技术研究计划实验动物项目(LGD20H160003);浙江省基础公益研究计划项目(LGF21H160010);国家卫生健康委科学研究基金-浙江省卫生健康重大科技计划重点项目(WKJ-ZJ-2117)。

摘　　要：目的:探讨胰岛素样生长因子2信使RNA(mRNA)结合蛋白3(IGF2BP3)对弥漫型胃癌细胞增殖、迁移和替代活化巨噬细胞(M2)巨噬细胞极化的影响。方法:本研究使用的细胞来源于浙江美森细胞和上海中国科学院细胞库。使用短发夹RNA慢病毒和慢病毒包装质粒分别感染人弥漫型胃癌细胞系NUGC-4和SNU-668,构建IGF2BP3敲低和过表达细胞株以及相应的对照细胞株。应用逆转录定量聚合酶链反应(RT-qPCR)和蛋白质印迹法(Western blot)分析各弥漫型胃癌细胞系的IGF2BP3表达水平和转染效率。通过细胞计数试剂盒-8(CCK-8)试验、集落形成试验和划痕试验,探究IGF2BP3对人弥漫型胃癌细胞的增殖和迁移能力的影响。使用佛波酯将人单核细胞THP-1诱导成巨噬细胞,收集对照组和实验组胃癌细胞的条件培养基(CM)处理巨噬细胞,通过RT-qPCR检测M2巨噬细胞标志物分化簇(CD)163、CD206、转化生长因子-β1(TGF-β1)、白细胞介素10(IL-10)和过氧化物酶体增殖激活受体γ(PPARG)。两组间均值比较采用t检验。结果:CCK-8试验显示,对照组450 nm吸光度(A450)高于IGF2BP3敲低组(24 h:0.22±0.10比0.10±0.09,t=6.806,P<0.05;48 h:0.46±0.21比0.30±0.10,t=8.372,P<0.05;72 h:0.71±0.38比0.51±0.11,t=7.827,P<0.05;96 h:1.01±0.44比0.79±0.09,t=4.539,P<0.05);而对照组A450低于IGF2BP3过表达组(24 h:0.25±0.02比0.38±0.01,t=12.074,P<0.05;48 h:0.60±0.02比0.87±0.04,t=9.273,P<0.05;72 h:0.93±0.02比1.36±0.02,t=27.955,P<0.05;96 h:1.40±0.05比1.88±0.08,t=9.057,P<0.05)。集落形成试验结果显示,对照组集落形成率高于IGF2BP3敲低组[(74.73±7.92)%比(52.90±6.25)%,t=3.747,P<0.05];对照组集落形成率低于IGF2BP3过表达组[(23.57±3.96)%比(41.47±3.26)%,t=6.040,P<0.05]。划痕试验显示,对照组划痕愈合率与IGF2BP3敲低组的差异无统计学意义[(3.42±1.68%)%比(2.33±1.66%)%,t=0.802,P>0.05]。对照CM处理组M2巨噬细胞标志物mRNA相对表达量高于IGF2BP3敲低CM处理组Objective To investigate the impact of insulin-like growth factor-2 mRNA(mRNA)-binding protein 3(IGF2BP3)on the proliferation,migration of diffuse gastric cancer cells,and the polarization of alternatively activated macrophages(M2).Methods The cells utilized in this study were sourced from the Zhejiang Meisen Cell Repository and the Shanghai Cell Bank of the Chinese Academy of Sciences.We employed short hairpin RNA lentivirus and lentiviral packaging plasmids to infect the human diffuse gastric cancer cell lines NUGC-4 and SNU-668.This approach was used to establish cell lines with knockdown and overexpression of IGF2BP3,along with their respective control cell lines.The expression levels of IGF2BP3 and the transfection efficiency in each diffuse gastric cancer cell line were analyzed using reverse transcription quantitative polymerase chain reaction(RT-qPCR)and Western blotting.To investigate the effects of IGF2BP3 on the proliferative and migratory capabilities of diffuse gastric cancer cells,we conducted assays including the Cell Counting Kit-8(CCK-8),colony formation,and scratch wound healing tests.To induce macrophage differentiation from human monocytes THP-1,phorbol myristate acetate was used.Conditioned media(CM)from control and experimental gastric cancer cells were collected and used to treat macrophages.The expression of M2 macrophage markers,including cluster of differentiation 163(CD163),CD206,transforming growth factor beta 1(TGF-β1),interleukin 10(IL-10),and peroxisome proliferator activated receptor gamma(PPARG),was assessed using RT-qPCR.Student’s t-test was employed for the comparison of mean values between the two groups.Results The CCK-8 assay revealed that the absorbance at 450 nm(A450)of the control group was significantly higher than that of the IGF2BP3 knockdown group at various time points(24 h:0.22±0.10 vs.0.10±0.09,t=6.806,P<0.05;48 h:0.46±0.21 vs.0.30±0.10,t=8.372,P<0.05;72 h:0.71±0.38 vs.0.51±0.11,t=7.827,P<0.05;96 h:1.01±0.44 vs.0.79±0.09,t=4.539,P<0.05).Conversely,the A45

关 键 词：胃癌 增殖 迁移 巨噬细胞 

分 类 号：R735.2[医药卫生—肿瘤]